A real-time, fluorescence-based assay for Rho-associated protein kinase activity

作者：Kelly Maia I; Bechtel Tyler J; Reddy D Rajasekhar; Hankore Erome D; Beck Jon R; Stains Cliff I^*

来源：Analytica Chimica Acta, 2015, 891: 284-290.

DOI：10.1016/j.aca.2015.07.058

• Kinase activity assay
• Fluorescence-based biosensor
• Rho-associated protein kinase
• Inhibition
• Small molecule screening

Inhibitors of Rho-associated protein kinase (ROCK) enzymatic activity have been shown to reduce the invasive phenotype observed in metastatic hepatocellular carcinoma (HCC). We describe the design, synthesis, and evaluation of a direct probe for ROCK activity utilizing a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox. The Sox fluorophore undergoes an increase in fluorescence upon phosphorylation of a proximal amino acid via chelation-enhanced fluorescence (CHEF, ex. = 360 nm and em. = 485 nm), allowing for the direct visualization of the rate of phosphate addition to a peptide substrate over time. Our optimal probe design, ROCK-S1, is capable of sensitively reporting ROCK activity with a limit of detection of 10 pM and a high degree of reproducibility (Z'-factor = 0.6 at 100 pM ROCK2). As a proof-of-principle for high-throughput screening (HTS) we demonstrate the ability to rapidly assess the efficacy of a 78 member, small molecule library against ROCK2 using a robotics platform. We identify two previously unreported ROCK2 inhibitor scaffolds, PHA665752 and IKK16, with IC50 values of 3.6 mu M and 247 nM respectively. Lastly, we define conditions for selectively monitoring ROCK activity in the presence of potential off-target enzymes (PKC alpha, PKA, and PAK) with similar substrate specificities.

• 出版日期2015-9-3